Disposable Fluid Circuits and Methods for Cell Washing

ABSTRACT

Systems and methods for the washing and processing of biological fluid/biological cells are disclosed. The systems and methods utilize a disposable fluid circuit including a spinning membrane separation device to wash the biological cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/537,856, filed Sep. 22, 2011, U.S. Provisional PatentApplication No. 61/618,307, filed Mar. 30, 2012, and U.S. ProvisionalPatent Application No. 61/636,411, filed Apr. 20, 2012, the contents ofeach are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present disclosure is generally directed to systems and methods forwashing biological cells. More particularly, the present disclosure isdirected to the sterile sequential processing of biological fluid andwashing of biological cells using one or a series of disposable fluidcircuits and a reusable processing apparatus in a closed system orenvironment.

BACKGROUND

The processing of biological fluid such as blood or blood componentstypically involves using a reusable processing apparatus (“hardware”)and a disposable fluid circuit adapted for mounting or other associationwith the reusable apparatus. The fluid circuit typically includes(plastic) bags and associated tubing that defines a flow path throughthe circuit. The disposable fluid circuit may also include one or moreseparation devices where the biological fluid/cells can be separatedinto two or more components, washed or otherwise processed.

The disposable fluid circuits typically include plastic containers andtubes that are pre-connected, pre-assembled, and pre-sterilized, such asby radiation or steam sterilization. In some processing systems andmethods, containers including liquids such as anticoagulant, saline,wash solution, storage media, or treating agents may likewise bepre-attached to the disposable fluid circuit, thereby creating a“closed” system. A “closed” system is one where the interior of thesystem, i.e., internal flow paths, separation chambers, etc., are notexposed or “opened” to the outside environment.

However, for a variety of reasons (e.g., sterilization incompatibility,timing of the different phases of the processing methods, sequence ofprocessing and/or treating steps), not all such liquids may bepre-attached to the disposable fluid circuit, in certain, more complexbiological fluid processing systems and methods, treating agents orother fluids necessary in the treatment of a given biological fluid orbiological cell product may require separate attachment to thedisposable fluid circuit at the time of use. In addition, in such morecomplex biological fluid processing systems and methods, two or morefluid circuits may be used in sequence to carry out the processingand/or treatment, and products collected using one circuit may need tobe connected to a second circuit while maintaining sterility of theoverall process.

Thus, it would be desirable to provide a series of fluid circuits thatallow for the sequential, sterile (i.e., in a “closed” or functionallyclosed system) processing of a biological fluid and/or desiredbiological cell population or product. More particularly, it would bedesirable to provide a series of disposable fluid circuits which arecompatible with one another and allow for sterile connection of selectedcontainers from one circuit to another circuit, as well as to certainauxiliary container processing sets. Finally, it would be desirable toprovide for a series of fluid circuits that are compatible with andadapted for sequential use with a single reusable apparatus. Thereusable apparatus may be pre-programmed to allow for the automatedprocessing of biological fluid and/or biological cell product with eachof the circuits of the series of disposable fluid circuits, as well aswith any auxiliary container sets.

SUMMARY

In one aspect, a method for biological cell washing is disclosed. Themethod includes obtaining a device that has a relatively rotatablecylindrical housing and internal member. The cylindrical housing has aninterior surface and the internal member has an exterior surface, thesurfaces defining a gap therebetween. At least the internal surface ofthe cylindrical housing or the external surface of the internal memberincludes a porous membrane. The porous membrane has a pore sizesufficient to separate biological cells from a liquid medium.

In accordance with the method described herein, a cell suspension, suchas biological cells suspended in a liquid medium, is introduced into thegap. The cell suspension to be washed may be provided in a sterile,disposable product bag, which is in flow communication with the spinningmembrane separator.

The cell suspension and wash medium are introduced into the gap and atleast one or both the cylindrical housing and internal member arerotated. As a result, the biological cells are separated from the liquidmedium in which they were suspended and are removed from the gap throughan outlet. The liquid medium, separated from the cells, is removed fromthe spinning membrane separator through a second outlet.

In another aspect, the present disclosure is directed to a disposable,fluid processing set useful in the washing of cells. The disposable setincludes a connector for establishing flow communication with a washsolution and a separator including a relatively rotatable housing andmembrane. The housing and membrane define a processing gap therebetween.The separator further includes at least one inlet and one outlet. Thedisposable set further includes an in-process container that includes aninlet and an outlet wherein the inlet of the in-process container is inopenable flow communication with the separator. The set also includes afinal product bag in openable flow communication with the outlet of theseparator.

In one more particular aspect, a system for the automated and sterilesequential treatment of biological fluid is provided. The systemincludes a reusable cell processing apparatus with a separator elementfor receiving a separation device and effecting the separation of thebiological fluid into two or more components. The system includes atleast a first disposable fluid circuit including at least one separationdevice in fluid communication with a first product container, and anaccess device for sterile connection to a source of biological fluid.The system includes at least a second disposable fluid circuit thatincludes at least one separation device in fluid communication with asecond product container, and an access device for sterile connection toa source of biological fluid.

In another aspect, the present disclosure is directed to a method forthe sterile sequential treatment of a biological fluid includingbiological cells. The method includes obtaining a reusable cellprocessing apparatus that includes a separator element that receives aseparation device for effecting the separation (and washing) ofbiological cells into two or more fractions; obtaining a firstdisposable fluid circuit that includes a separation device in fluidcommunication with a pre-attached first product container and an accessdevice for sterile connection to a source of biological fluid; mountingthe first disposable processing circuit onto the reusable cellprocessing apparatus; sterilely joining a source of biological fluid tothe first disposable fluid circuit; introducing the biological fluidinto the separation device to separate a desired biological cell productfrom a remainder of the biological fluid; collecting the separatedbiological cell product in the product container; transferring thecollected product to a pre-attached separation chamber; removing thefirst disposable processing set from the reusable cell processingcircuit; sterilely transferring the product of the detached firstproduct container to a second disposable cell processing circuit, thesecond disposable processing circuit including a separation device influid communication with a pre-attached second product container and anaccess device for sterile connection to the source of biological fluid;mounting the second disposable processing circuit onto the reusable cellprocessing apparatus; introducing the contents of a source containerinto the separation device to separate a desired biological fluidcomponent from a remainder of said biological fluid; and collecting adesired separated biological fluid component in the second productcontainer of the second disposable fluid circuit.

In a further aspect, the systems and methods disclosed herein mayinclude further sequential processing using additional disposable fluidcircuits (e.g., third, fourth) as well as auxiliary container sets.

In another aspect, a method for introducing a biological agent into aclosed processing system such as, but not limited to, the systemsdescribed herein is also disclosed. The method includes providing aclosed processing system that includes a container that holds an amountof a cell population. The method also includes filling, in a controlledenvironment, a delivery device with a desired amount of a treatingagent. A container of a carrier solution is provided and the agent iscombined with carrier solution in the container in a controlledenvironment. The container of carrier solution/treating agent is thenremoved from the controlled environment and joined to the container ofthe processing system in a sterile fashion.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic view of one embodiment of a disposable fluidcircuit useful in the systems and methods described herein;

FIG. 2 is a schematic view of another embodiment of a disposable fluidcircuit useful in the systems and methods described herein;

FIG. 3 is a schematic view of yet another embodiment of a disposablefluid circuit useful in the systems and methods described herein;

FIG. 4 is a schematic view of still another embodiment of a disposablefluid circuit useful in the systems and methods described herein;

FIG. 5 is an enlarged view of the front panel of the reusable processingapparatus;

FIG. 6 is a perspective view of a separation/washing device using aspinning membrane;

FIG. 7(a) is a perspective view, partially broken away, of theseparation/washing of FIG. 6;

FIG. 7(b) is a cross-sectional view of the separation device of FIG.7(a);

FIG. 8 is a schematic view of an auxiliary container set for use incombination with one or more of the disposable fluid circuits of FIGS.1-4;

FIG. 9 is a schematic view of a further auxiliary container set for usewith one or more of the disposable fluid circuits of FIGS. 1-4;

FIGS. 10(a)-10(n) are flow diagrams showing the method steps in oneexemplary method of biological cell processing using the reusableprocessing apparatus and the series of disposable fluid circuitsdisclosed herein;

FIGS. 11(A)-(C) show the steps of providing a treating agent and carriersolution to a disposable fluid circuit in a sterile manner;

FIG. 12 diagrammatically shows the reduction in supernatant content inthe biological fluid and cells washed in accordance with the methods andsystems disclosed herein;

FIG. 13 diagrammatically shows the further reduction in supernatantcontent in the biological fluid and cells washed in accordance with themethods and systems disclosed herein;

FIG. 14 is another view of the front panel of a reusable processingand/or cell washing apparatus with a disposable fluid circuit loadedthereon; and

FIG. 15 depicts a pair of graphs showing the cell retention using twodifferent membrane materials for a spinning membrane device.

DETAILED DESCRIPTION

Systems and methods for the automated sequential sterile processing ofbiological fluid are disclosed herein. The systems disclosed typicallyinclude a reusable separation apparatus and one or more disposableprocessing circuits adapted for association with the reusable apparatus.The reusable separation apparatus may be any apparatus that can providefor the automated processing of biological fluid. By “automated,” it ismeant that the apparatus can be pre-programmed to carry out theprocessing steps of a biological fluid processing method withoutsubstantial operator involvement. Of course, even in the automatedsystem of the present disclosure, it will be understood that someoperator involvement will be required, including the loading of thedisposable fluid circuits and entering processing parameters. Additionalmanual steps may be required as well. However, the reusable apparatuscan be programmed to process biological fluid through each of thedisposable circuits described below without substantial operatorintervention.

The reusable processing apparatus is typically capable of effecting theseparation of a biological fluid that includes biological cells into twoor more components or fractions. Thus, the reusable apparatus maygenerate conditions which allow for the separation of a biological fluidinto selected components or fractions. In accordance with the presentdisclosure, one preferred means for separating biological fluid into itsconstituent components or fractions is an apparatus that uses a spinningporous membrane to separate one component from other components. Anexample of such apparatus is the Autopheresis C® sold by Fenwal, Inc. ofLake Zurich, Ill. A detailed description of a spinning membrane may befound in U.S. Pat. No. 5,194,145 to Schoendorfer, which is incorporatedby reference herein in its entirety, and in International (PCT)Application No. PCT/US2012/028492, filed Mar. 9, 2012, the contents ofwhich is also incorporated herein in its entirety. In addition, systemsand methods that utilize a spinning porous membrane are also disclosedin U.S. Provisional Patent Application No. 61/537,856, filed on Sep. 22,2011, and International (PCT) Application No. PCT/US2012/028522, filedMar. 9, 2012, the contents of each are incorporated herein by reference.The references identified above describe a membrane covered spinnerhaving an interior collection system disposed within a stationary shell.While a detailed discussion of the separation device is beyond the scopeof this application, the spinning membrane separation device is shown inFIGS. 6, 7(a)-7(b) and is discussed below. In another embodiment, thereusable apparatus may generate a centrifugal field to effectseparation.

Turning now to FIGS. 1-4, the systems described herein preferablyinclude two or more disposable fluid circuits for use in the processingof biological fluid. While the circuits described herein may be used asstand alone circuits, more preferably, at least two or more disposablefluid circuits are used in combination and in series for the separation,washing, volume reduction and/or other processing of a biological fluid.As will be apparent from the description and figures below, the circuitsused herein share many common elements and as such, where appropriate,identical reference numbers are generally used throughout to refer toidentical or substantially identical elements of each of the circuits100, 100′, 100″, and 100′″. For example, the circuits 100, 100′, 100″,and 100′″ described below may include an integrated separation device,such as, but not limited to, the spinning membrane 101 (e.g., 101′,101″, and 101′″) described above. Circuits 100, 100′, 100″, and 100′″may also include waste container 140, product container 150, andin-process container 122. Disposable fluid circuits of the typedescribed below may further include sampling assemblies 112 and 152 forcollecting samples of source biological fluid, “final” product, or otherintermediate products obtained during the biological fluid processing.

As will be seen in the Figures and described in greater detail below,the disposable fluid processing circuits include tubing that definesflow paths throughout the circuits, as well as access sites for sterileor other connection to containers of processing solutions, such as washsolutions, treating agents, or sources of biological fluid. As shown inFIGS. 1-4, the tubing of circuits 100, 100′, 100″, 100′″ includes spacedtubing segments identified by reference numerals 162, 166, 168 (andcounterpart reference numeral 162′, 162″, etc.). The tubing segments areprovided for mating engagement with the peristaltic pumps of thereusable hardware apparatus 200 discussed below. The containers and theplastic tubing are made of conventional medical grade plastic that canbe sterilized by sterilization techniques commonly used in the medicalfield such as, but not limited to, radiation or autoclaving. Plasticmaterials useful in the manufacture of containers and of the tubing inthe circuits disclosed herein include plasticized polyvinyl chloride.Other useful materials include acrylics. In addition, certainpolyolefins may also be used.

As will be apparent from the disclosure herein, source containers may beattached in sterile fashion to each of the circuits 100, 100′, 100″, and100′″. Source containers 102 for connection to one disposable circuitmay be the product containers 150 of another circuit used in an earlierstep of the overall method of processing. Alternatively, the contents ofa product container 150 may be further processed or separated and thentransferred in sterile fashion to the source container 102 of alater-in-series fluid circuit.

The biological cell suspension to be washed or otherwise treated istypically provided in a source container 102, shown in FIG. 1 as(initially) not connected to the disposable set. As noted above, sourcecontainer 102 may be attached (in sterile fashion) at the time of use.Source container 102 has one or more access sites 103, 105, one of whichmay be adapted for (sterile) connection to fluid circuit 100 at dockingsite 104. Preferably, source containers may be attached in a sterilemanner by employing sterile docking devices, such as the BioWelder,available from Sartorius AG, or the SCD IIB Tubing Welder, availablefrom Terumo Medical Corporation. A second access port 105 may also beprovided for extracting fluid from the source container 102.

As further shown in FIG. 1, tubing segment 106 extends from docking site104 and may optionally include a sampling sub-unit at branched-connector108. One branch of branched-connector 108 may include a flow path 110leading to sampling assembly 112. Sampling assembly 112 allows for thecollection of a sample of the incoming source fluid. Flow to thesampling assembly 112 is typically controlled by clamp 114. The otherbranch of branched-connector 108 is connected to and in flowcommunication with tubing 116. Tubing 116 is connected to furtherdownstream branched-connector 118. Branched-connector 118 communicateswith tubing 116 and tubing 120, which provides a fluid flow path from“in-process” container 122, described in greater detail below. Tubingsegment 124 extends from branched-connector 118 and is joined to a portof further downstream branched-connector 126. A separate flow pathdefined by tubing 128 is also connected to a port of branched-connector126.

In accordance with the fluid circuit of FIG. 1, a container of wash orother processing/treating solution may be attached (or pre-attached) toset 100. As shown in FIG. 1, tubing 132 (defining a flow path)preferably includes and terminates in an access site such as spikeconnector 134. Access site 134 is provided to establish flowcommunication with a container 135 (shown in FIG. 14) of a wash fluid,such as saline or other solution. Tubing 128 may include an in-linesterile barrier filter 130 for filtering any particulate from a fluidbefore it enters the flow path leading to second branched-connector 126and, ultimately separator 101. In one embodiment, sterile barrier filtermay be a 0.2 μm filter. The wash medium or fluid flows from the washfluid source through tubing segment 132, where it is filtered by thesterile barrier filter 130 described above, and then passes throughtubing 128 to the input of the branched-connector 126 described above.

Tubing segment 136 defines a flow path connected at one end tobranched-connector 126 and to an inlet port 20 of the separator 101.Preferably, in accordance with the present disclosure, separation device101 is a spinning membrane separator of the type described in U.S. Pat.No. 5,194,145 and U.S. Pat. No. 5,053,121, which are incorporated byreference, U.S. Provisional Patent Application Ser. No. 61/451,903 andPCT/US2012/028522, also previously incorporated herein by reference.

As shown in FIG. 1 (and described in greater detail in connection withFIGS. 6, 7(a)-7(d), the spinning membrane separator 101 has at least twooutlet ports. Outlet 46 of separator 101 receives the waste from thewash (i.e., the diluted suspension medium) and is connected to tubing138, which defines a flow path to waste product container 140. The wasteproduct container includes a further connection port 141 for sampling orwithdrawing the waste from within the product container.

Separation device 101 preferably includes a second outlet 48 that isconnected to tubing segment 142 for directing the desired biologicalcell/fluid product to “final” product container. The other end of tubingsegment 142 is connected to branched-connector 144, which branches intoand defines a flow path to one or more in-process containers 122 and aflow path to a final product container 150, The final product container150 may also include a sampling assembly 152. Flow control to thesampling assembly 152 is preferably controlled by clamp 156. The flowpath through the access port 154 is controlled by clamp 158.

As further shown in FIG. 1, depending on the processing method and thebiological fluid or biological cells being processed, fluid circuit 100may optionally include an additional chamber for the processing and/orfurther separation of the biological fluid or cells. As shown in FIG. 1,fluid circuit 100 includes an additional chamber 160 for processing thecontents in “final” product container 150. Chamber 160 may be acentrifugal bowl or channel integrally connected to circuit 100.Alternatively, chamber 160 may use a different separation principle(i.e., other than centrifugation) to effect the desired processing ofthe biological fluid or cells introduced therein. Chamber 160 mayinclude one or more ports 162 and 164 for establishing fluidcommunication with product container 150 or other container(s) used inthe method of processing. For example, port 164 is connected to tubingsegment 166 which defines a flow path terminating in access site 168.Access site 168 may be a conventional spike or similar access deviceadapted for accessing a port of a fluid container including a treatingor processing agent. Where access site 168 is a conventional spike, theflow path defined by tubing segment 166 may further include asterilizing filter 172. Alternatively, access site 168 may be adaptedfor sterile connection in the manner previously described.

As noted above, chamber 160 is integral with disposable fluid circuit100 and allows for further processing of the fluid/cells collected inproduct container 150. In one embodiment, chamber 160 may be a bowl orother container adapted for use with a centrifuge device. An example ofsuch a chamber is provided in U.S. Pat. No. 5,663,051, the contents ofwhich are incorporated herein by reference. Chamber 160 may bedisconnected from circuit 100, placed inside a centrifuge device, andsubjected to a centrifugal field where the biological fluid/cells may beseparated into desired components or fractions.

Turning now to FIG. 2, disposable fluid circuit 100′ includes many ofthe same elements and is substantially similar to fluid circuit 100 ofFIG. 1. Thus, for example, fluid circuit 100′ includes a separationdevice 101′, waste container 140′, final product container 150′,in-process container 152′, and an added separation chamber 160′, asshown and described above. Disposable fluid circuit 100′ also includes asampling assembly 152′ between separation chamber 160′ and productcontainer 150′. Tubing and access sites are also provided substantially,as shown, with respect to the disposable fluid circuit 100 of FIG. 1.

Turning now to FIG. 3, a further disposable fluid circuit 100″ is alsoshown. Again, as with the disposable fluid circuit of FIG. 2, circuit100″ likewise includes many of the same elements, connections, accesssites, sampling assemblies and containers, as previously described withrespect to circuits 100 and 100′. As shown in FIG. 3, in one embodiment,fluid circuit 100″ is devoid of separation chamber 160 or 160′. Instead,final product container 150″ may include a port with a tube extendingtherefrom terminating in a docking site for attachment to anotherauxiliary container set or other containers used in the method ofprocessing biological fluid. In addition, fluid circuit 100″ may includedual access sites 134 a″ and 134 b″. Dual access sites are optional andmay be provided for the addition of selected carrier and/or washsolutions in connection with one method of processing. Fluid processingcircuit 100″ may also include an empty source container 102″, whichincludes a tubing extending from port 103 and terminating in a steriledocking site 103″.

FIG. 4 shows a further disposable fluid circuit 100′″, which may also beused in connection with and in conjunction with, or as part of a seriesof disposable fluid circuits 100, 100′ and 100″, in accordance with amethod for processing biological fluid and/or biological cells. Fluidcircuit 100′″ likewise includes many of the same elements as the earlierfluid circuits 100, 100′, 100″, which will not be repeated here. Thepurpose and function of the various elements will become apparent inconnection with the description of an exemplary method of processingbiological fluid and/or biological cells set forth below.

FIG. 5 shows the front panel 201 of reusable hardware processingapparatus 200. Apparatus 200 may be of compact size suitable forplacement on a table top of a lab bench and adapted for easy transport.Alternatively, apparatus 200 may be supported by a pedestal that can bewheeled to its desired location. In any event, as shown in FIG. 5,apparatus 200 includes a plurality of peristaltic pumps such as pumps202, 204 and 206 on front panel 201. Pump segments of the disposablefluid circuit (described above) are selectively associated withperistaltic pumps 202, 204, and 206. The peristaltic pumps articulatewith the fluid sets of FIGS. 1-4 at the pump segments identified byreference numerals 162, 166, 168 and advance the cell suspension orother fluid within the disposable set, as will be understood by those ofskill in the art. Apparatus 200 also includes clamps 210, 212, 214, and216. Clamps 210, 212, 214, and 216 are used to control the flow of thecell suspension through different segments of the disposable set, asdescribed above.

Apparatus 200 also includes several sensors to measure variousconditions. The output of the sensors is utilized by device 200 tooperate one or more wash or processing cycles. One or more pressuretransducer sensor(s) 226 may be provided on apparatus 200 and may beassociated with a disposable set “100” at certain points to monitor thepressure during a procedure. Pressure transducer 226 may be integratedinto an in-line pressure monitoring site (at, for example, tubingsegment 136), to monitor pressure inside separator 101. Air detector 238sensor may also be associated with the disposable set 100, as necessary.Air detector 238 is optional and may be provided to detect the locationof fluid/air interfaces.

Apparatus 200 includes weight scales 240, 242, 244, and 246 from whichthe final product container, in-process container, source container, andany additional container(s), respectively, may depend and be weighed.The weights of the bags are monitored by weight sensors and recordedduring a washing or other procedure. From measurements of the weightsensors, the device determines whether each container is empty,partially full, or full and controls the components of apparatus 200,such as the peristaltic pumps and clamps 210, 212, 214, 216, 218, 220,222, and 224.

Apparatus 200 includes at least one drive unit or “spinner” 248, whichcauses the indirect driving of the spinning membrane separator 101(101′, 101″ or 101′″). Spinner 248 may consist of a drive motorconnected and operated by apparatus 200, coupled to turn an annularmagnetic drive member including at least a pair of permanent magnets. Asthe annular drive member is rotated, magnetic attraction betweencorresponding magnets within the housing of the spinning membraneseparator cause the spinner within the housing of the spinning membraneseparator to rotate.

Turning to FIGS. 6, 7(a) and 7(b), a spinning membrane separationdevice, generally designated 101, is shown. Such a device 10 forms partof each of the disposable circuits 100, 100′, 100″ and 100′″.

Device 101 includes a generally cylindrical housing 12, mountedconcentrically about a longitudinal vertical central axis. An internalmember 14 is mounted concentric with the central axis 11. Housing 12 andinternal member 14 are relatively rotatable. In the preferredembodiment, as illustrated, housing 12 is stationary and internal member14 is a rotating spinner that is rotatable concentrically withincylindrical housing 12, as shown by the thick arrow in FIG. 6. Theboundaries of the blood flow path are generally defined by gap 16between the interior surface of housing 12 and the exterior surface ofrotary spinner 14. The spacing between the housing and the spinner issometimes referred to as the shear gap. In one non-limiting example, theshear gap may be approximately 0.025-0.050 inches (0.067-0.127 cm) andmay be of a uniform dimension along axis 11, for example, where the axisof the spinner and housing are coincident. The shear gap may also varycircumferentially for example, where the axis of the housing and spinnerare offset.

The shear gap also may vary along the axial direction, for examplepreferably an increasing gap width in the direction. Such a gap widthmay range from about 0.025 to about 0.075 inches (0.06-0.19 cm). The gapwidth could be varied by varying the outer diameter of the rotor and/orthe inner diameter of the facing housing surface. The gap width couldchange linearly or stepwise or in some other manner as may be desired.In any event, the width dimension of the gap is preferably selected sothat at the desired relative rotational speed, Taylor-Couette flow, suchas Taylor vortices, are created in the gap.

Biological fluid is fed from an inlet conduit 20 through an inletorifice 22, which directs the fluid into the fluid flow entrance regionin a path tangential to the circumference about the upper end of thespinner 14. At the bottom end of the cylindrical housing 12, the housinginner wall includes an exit orifice 34.

Cylindrical housing 12 is completed by an upper end cap 40 having an endboss 42, the walls of which are nonmagnetic, and a bottom end housing 44terminating in a outlet orifice 46 concentric with the central axis.

With reference to FIGS. 7(a) and 7(b), spinner 14 is rotatably mountedbetween upper end cap 40 and the bottom end housing 44. Spinner 14comprises a shaped central mandrel or rotor 50, the outer surface ofwhich is shaped to define a series of spaced-apart circumferentialgrooves or ribs 52 separated by annular lands 54. The surface channelsdefined by the circumferential grooves 52 are interconnected bylongitudinal grooves 56. At each end of the mandrel 50, these grooves 56are in communication with a central orifice or manifold 58. In theillustrated embodiment, the surface of the rotary spinner 14 is at leastpartially, and is preferably substantially or entirely, covered by acylindrical porous membrane 62. The membrane 62 typically has a nominalpore size of 0.6 microns, but other pore sizes may alternatively beused. Membranes useful in the washing methods described herein may befibrous mesh membranes, cast membranes, track-etched membranes or othertypes of membranes that will be known to those of skill in the art. Forexample, in one embodiment, the membrane may have a polyester mesh(substrate) with nylon particles solidified thereon, thereby creating atortuous path through which only certain sized components will pass. Inan embodiment, the nylon membrane may have a pore size of approximately0.65 μm and a thickness of approximately 100 μm or greater. Membranes ofthis type will typically retain all cellular components (e.g., red bloodcells, white blood cells) and certain formed blood components, e.g.,platelets. In another embodiment, the membrane may be made of a thin(approximately 10-15 micron (μm) thick) sheet of, for example,polycarbonate. In this embodiment, pores (holes) may be cylindrical andlarger than those described above. For example, pores may beapproximately 3-5 microns (μm), and more preferably about 4 μm. Thepores may be sized to allow small formed components (e.g., platelets,microparticles, etc.) to pass, while the desired cells (e.g., whiteblood cells and larger red blood cells) are collected. FIG. 15graphically illustrates cell retention with a nylon membrane and apolycarbonate membrane as described above. (The abbreviation “LOD” inFIG. 15 refers to “limits of detection.”)

Device 10 is mounted in the upper end cap to rotate about a pin 64,which is press fit into the end cap 40 on one side and seated within acylindrical bearing surface 65 in an end cylinder 66 forming part of therotary spinner 14. The internal spinner 14 or outer housing 12 may berotated by any suitable rotary drive device or system. As illustrated,the end cylinder 66 is partially encompassed by a ring 68 of magneticmaterial utilized in indirect driving of the spinner 14. A drive motor70 exterior to the housing 12 is coupled to turn an annular magneticdrive member 72 that includes at least a pair of interior permanentmagnets 74. As the annular drive member 72 is rotated, magneticattraction between the ring 68 interior to the housing 12 and themagnets 74 exterior to the housing locks the spinner 14 to the exteriordrive, causing the spinner 14 to rotate.

At the lower end of the rotary spinner 14, the central outlet orifice 58communicates with a central bore 76 in an end bearing 78 that isconcentric with the central axis. An end bearing seat is defined by aninternal shoulder 80 that forms a lower edge of a central opening 82.The central opening 82 communicates with the outlet orifice 46. If theinner facing surface of the housing is covered entirely or partially bya membrane, a fluid collection or manifold may be provided beneath themembrane to collect a blood fraction and direct it through a housingoutlet (not shown).

U.S. Provisional Patent Application No. 61/537,856, filed on Sep. 22,2011, the contents of which are incorporated herein by reference, andInternational Application No. PCT/US2012/028522, filed Mar. 9, 2012, thecontents of which are also incorporated herein by reference, disclosemethods and systems for washing biological cells using a reusablehardware apparatus and disposable fluid circuit including a spinningmembrane separator.

FIGS. 10(a)-10(n) diagrammatically set forth one exemplary andnon-limiting method of cell processing (e.g., washing) using adisposable fluid circuit and reusable hardware of the type discussedabove. The exemplary method involves the processing, washing, treatingand incubating of biological cells, such as mononuclear cells forsubsequent therapeutic administration. It will be understood, however,that the method described below is not intended to limit the inventionor the use of the system and the fluid circuits described below. Othermethods using less than all of the disposable fluid circuits and/orauxiliary container sets, or processing circuits that have beenmodified, or fewer than all of the enumerated steps may be practicedwithout departing from the spirit or scope of the present invention.

Many of the steps described below are performed by the software drivenmicroprocessing unit of apparatus 200 with certain steps performed bythe operator, as noted. Turning first to FIG. 10(a), the apparatus 200is switched on at step 300. Apparatus 200 conducts self-calibrationchecks 302, including the checking of the peristaltic pumps, clamps, andsensors. Apparatus 200 then prompts the user to enter selectedprocedural parameters (step 304), such as the washing procedure to beperformed, the amount of cell suspension to be washed, the number ofwashings to take place, etc. The operator may then select and enter theprocedural parameters for the wash procedure (step 306).

Apparatus 200 (through the controller) confirms the parameter entry 306and then prompts the operator to load (step 310) the disposable set. Theoperator then loads the disposable set (step 312) onto the panel ofapparatus 200. In one exemplary embodiment, the disposable set may bethe fluid circuit of FIG. 1. After installation of the disposable set,apparatus 200 confirms installation as shown in (step 314).

After the disposable set is mounted, apparatus 200 automatically checksto determine whether the disposable set is properly installed (step316). After apparatus 200 determines that the disposable set is properlyinstalled, the controller prompts the operator to connect the biologicalfluid and wash medium (step 318). The operator then connects the washmedium (such as, but not limited to saline) (step 320) to the disposableset via a spike connector. The operator then connects source container102 of the biological fluid or biological cell product (typicallyderived from an earlier, separate procedure (step 322)) to thedisposable set via a spike connector or sterile connection as previouslydescribed. In one embodiment, the source of biological fluid/cells maybe apheresis-collected mononuclear cells.

As shown in FIG. 10(b), after the source of biological fluid and washmedium are connected to the disposable set, the operator confirms thatthe solutions are connected (step 324). The device prompts the operatorto take a cell suspension sample (step 326). The operator or the devicethen opens sampling assembly clamp 328 to introduce fluid into thesample chamber of the sampling assembly (step 340). Once the samplechamber is sufficiently filled, it is then sealed and removed (342) fromthe disposable circuit. The operator confirms (step 344) that a samplehas been taken. Following the removal of the sample chamber, thedisposable fluid circuit is primed (step 346) for the (initial) washprocess. In one embodiment, the circuit may be primed with saline,although other bio-compatible aqueous solutions may also be used.

The controller of separation apparatus then commences the wash process.The biological cells to be washed are transferred from source container(e.g., 102 of FIG. 1) through the disposable set to the spinningmembrane separator 101 via the operation of one or more peristalticpumps 202, 204 and 206. Likewise, the wash medium is delivered from itscontainer, through the disposable circuit to the spinning membraneseparator 101. In a preferred embodiment, the original cells of the cellsuspension are concentrated and/or collected in either an in-process bag(for further processing) or collected in a final product container 150,while supernatant is separated and removed to waste container 140. In apreferred embodiment, the process provides a final concentratedbiological cell product 150 resuspended in approximately 200 ml of thewash (e.g., saline) solution with approximately a 2 log reduction ofsupernatant contents. If (further) washing or diluting of the cellsuspension is necessary, the cell suspension in the in-process bag maybe washed (a second time) with the same or different wash mediumfollowing the process outlined above. Prior to the conclusion of eachwash cycle, the cell suspension volume or weight is measured andrecorded (step 350). When the concentration of the cells to wash mediumreaches an acceptable level the final product bag is filled.

As shown in FIG. 10(c), once the desired volume of the final product iscollected, the control and operation device prompts the operator tosample and seal the final product container (step 352). After sampling,the operator then seals and removes from the disposable circuit thewashed cell suspension in the final product container 150. The finalproduct container may then be agitated (step 354). The operator opensthe sample chamber by opening the clamp (step 356), and the samplechamber is allowed to fill (step 358). Once the sample chamber isfilled, the clamp is closed and the sample assembly is sealed andremoved (step 360). The operator then seals the disposable set lines(step 362) and confirms that the product container has been sealed andremoved, a sample assembly has been filled and removed, and that thedisposable set lines have been sealed 364. The control and operationdevice then prompts the operator to remove the disposable fluid circuit100, as shown in step 366. The operator then removes and discards thedisposable circuit 100 as shown in step 368. A “procedure wrap around,”as referenced in FIG. 10(d) (and elsewhere), refers to when theapparatus has completed one procedure and is ready for a new procedure,restarting at a given state.

As shown in FIG. 1, disposable fluid circuit 100 may include anadditional processing/separation chamber 160 integrally connected to thecircuit. Chamber 160 may be provided where further processing/separationof the washed product in product container 150 may be required as partof a cell treatment method. Thus, in accordance with one such method,chamber 160 may include an access site for sterile connection to asource of a treating or separation-enhancing agent such as a buoyantdensity solution (BDS). Such solution may be transferred prior to thesampling step described above. Once the sampling assembly has beenremoved, washed product from container 150 may be automaticallydispensed (step 370) into chamber 160. Once the washed product has beentransferred to separation chamber 160, the flow paths between container150 and chamber 160 may be sealed and chamber 160 may be detached fromthe remainder of circuit 100. Chamber 160 may then be subjected to acentrifugation step (in a separately provided centrifuge) where thewashed biological cell product/BDS suspension is separated into thedesired lighter and heavier fractions.

The desired fraction may then be decanted to a separate container (step371) that will serve as a source container in the further processing ofthe biological cells. For example, in one exemplary method, the lighterfraction in chamber 160 may be decanted to a source container 102′ shownin FIG. 2.

For further processing/washing of the contents of source container 102′,the system may again prompt the operator to enter the proceduralparameters (step 372), as shown in FIG. 10(d). The system may thenprompt the operator to load disposable circuit 100′ of FIG. 2 (step374). Once the operator has installed circuit 100′ (and confirmed itsinstallation), the system may prompt the operator to connect therequired solutions (step 376). The operator then connects the desiredwash solution (e.g., saline) at access site 134′ and sterilely connectssource container 102′ to the terminal end of flow path 106′. Aspreviously discussed, sterile connection may be achieved by employingsterile docking devices, such as the BioWelder, available from SartoriusAG, or the SCD IIB Tubing Welder, available from Terumo MedicalCorporation. Other methods of sterilely connecting source container 102′to fluid circuit 100′ may be also be used. The system is pre-programmedto prime circuit 100′ and then deliver the product from source container102′ and the wash solution to separation device 101′. Waste(supernatant) from the washing step is directed to waste container 140′and the desired cellular product is delivered to product container 150′.The volume/weight of container 150′, which is suspended from thesystem's weight scales (240-248 of FIG. 6) is recorded (step 378) by thesystem and the operator is then prompted to collect a sample of thewashed product.

Sampling may proceed substantially as described in connection with thesampling of washed “final” product in disposable fluid circuit 100. Inaddition, it will be noted that disposable fluid circuit 100′ may alsoinclude a pre-connected separation chamber 160′. Chamber 160′ mayinclude a flow path extending from an outer port and terminating in anaccess site for sterile connection to a source of a treating or selectedseparation-enhancing agent such as a buoyant density solution (BDS).Such solution may be transferred prior to or after (as shown in FIGS.10(f) and 10(g)) the sampling step described above. Once the samplingassembly has been removed, washed product from container 150′ may beautomatically dispensed (step 382) into chamber 160′. Once the washedproduct has been transferred to separation chamber 160′, the flow pathsbetween container 150′ and chamber 160′ may be sealed and chamber 160′may be detached from the remainder of circuit 100′. Chamber 160′ maythen be subjected to centrifugation or other separation step (in aseparately provided centrifuge) where the washed biological cellproduct/BDS suspension is separated into the desired lighter and heavierfractions.

Following separation of the cell suspension in separation chamber 160′,the desired fraction may then be decanted to a separate container thatwill serve as a source container in the further processing of thebiological cells or, more preferably, to an auxiliary container set. Anexample of an auxiliary container set is shown in FIG. 8. As shown, inFIG. 8, auxiliary container set 500 includes sterile dock access site502 and at least two containers 504 and 506 in openable flowcommunication with access site 502 and separated by branch member 508.In accordance with the one embodiment of a method of processing, washingand treating mononuclear cells, auxiliary container set 500 is sterilelyjoined to chamber 160′. One fraction (e.g., the lighter fraction) may bedecanted as waste into one of the containers (e.g., 504), while aresuspending medium (e.g., saline) may be introduced into chamber 160′to resuspend the remaining and desired biological cell product. Theauxiliary container set can be disconnected from chamber 160′ and thecontents of chamber 160′ may then be transferred to the next sourcecontainer 102″ shown in FIG. 3.

The system may then prompt the operator to enter the processingparameters (step 384 of FIG. 10(g)) for further processing and to loadfluid circuit 100″ (of FIG. 3) onto reusable processing apparatus 200(step 386). Once proper installation of the next in sequence fluidprocessing circuit has been confirmed, the system will prompt theoperator to connect the solutions (step 388), including wash solution(saline) at access site 134 a, a carrier solution at access site 134 b,and source container 102″.

In accordance with one example of a cell processing method (e.g.,mononuclear cell processing), the system may be programmed to deliverthe contents of source container 102″ to separation device 101″ with acarrier solution and saline (step 389). Separated biological cells witha carrier solution are collected in product container 150″.

As seen in FIG. 3, product container 150″ may also preferably includetwo access sites 154″ and 155″ for sterile docking. In a specificembodiment of a method for processing cells (such as mononuclear cells),access site 154″ may be sterile docked to a further auxiliary set 157,such as the one shown in FIG. 9. Auxiliary container set 157 (FIG. 9)may include one or more culture containers 159 a, b, c and d. Accesssite 155 may be adapted for sterile connection to a container oftreating agent 161 (such as an antigen or other agent useful in theculturing and preparation of selected cells) or, more preferably, acontainer of combined carrier solution and treating agent. In thispreferred embodiment, separate addition (and connection) of carriersolution (at access site 134 a of circuit 100″) and of treating agentcan be avoided by providing a container of carrier solution with anappropriate amount of antigen contained therein. This way the combinedcarrier solution and agent suspension can be sterile docked to accesssite 155 of product container 150″.

The treating agent/carrier solution may be prepared and delivered in thefollowing manner, as depicted in FIG. 11(A)-(C). In one embodiment, asyringe 460 or other delivery device may be used to remove a desiredamount of antigen from vial 462 in a controlled environment, such as aBiological Safety Cabinet (BSC) 464 or other similar hood or enclosure.The contents of filled syringe 460 may then be dispensed (still in theBSC or other controlled environment) into container 466 containing thecarrier solution. Container 466 may include a port 467 having apierceable septum which seals after penetration by the needle of syringe460. Container 466 preferably includes a sealed tubing 469 in openableflow communication with the chamber of container 466. Once the antigenor other agent has been combined with the carrier solution, container466 may be brought out from the BSC or other controlled environment andjoined to fluid circuit 100″ and more specifically product container150″ by connecting in sterile fashion (as described above by using asterile weld device, such as a Terumo SCD IIB welder) tube 469 andaccess site 155 of product container 150″. Once a sterile connection hasbeen made, a flow path between container 466 and container 150″ isestablished. Treating agent (in carrier liquid) may be delivered to thecells in container 150″ by gravity flow or by the action of a pump (notshown).

In either embodiment, system may prompt (step 390 in FIG. 10(i)) theoperator to sterilely connect auxiliary set 157, as well as prompt theoperator to sterilely connect the container (e.g., 466) of treatingagent 161 (step 392 in FIG. 10(j)). In a preferred embodiment, based onthe composition of the samples collected prior to wash step 389, thesystem disclosed herein may automatically calculate the amount ofproduct in container 150, the amount of carrier/agent to be added toeach of the culture containers, and/or if separately added, the amountof the treating agent (antigen) to be added to the culture containers159 a-d (step 394). After transfer, the operator may further be promptedto seal and remove set 159 and remove circuit 100 (step 396).

In a further processing step, after an appropriate incubation period,the contents of culture containers 159 a-d may be pooled in sourcecontainer 102′″ of the disposable fluid circuit 100′″ of FIG. 4 (step398, FIG. 10(I)). Specifically, the pooling container set 157 may besterilely connected to source container 102′″ of the disposable fluidcircuit 100′″. The operator may be further prompted to connectadditional wash solution and ultimate storage solution in step 400. Oncethe solutions are connected, the system will automatically process/washthe pooled contents of source container 102′″ in separation device101′″, removing supernatant and collecting the desired biological cellswith the storage solution (for example, Ringers lactate) in finalcontainer 150′″. The washing process preferably yields a concentratedcell product resuspended in approximately 295 ml of storage solutionwith approximately a 3.5 log reduction of supernatant. Samples of thecontents in final container 150′″ may be collected, as previouslydescribed and as set forth in FIG. 10(m). The contents of finalcontainer 150′″ may then be ready for therapeutic administration.

The systems and methods described herein are effective in the washing ofcells such as red blood cells and/or white blood cells. In one exampleof red cell washing, frozen red blood cells may be incubated within arejuvenating solution such as Rejuvesol. The solution may be steriledocked or otherwise included in the closed system of the disposableprocessing sets of the type described above. Incubation occurs atapproximately 37° C. within the closed system. The treated cells maythen be washed with a washing solution such as saline, Adsol or E-Sol(the latter of which are red cell storage solutions) to reconstitute thered blood cells for subsequent storage and transfusion.

The systems and methods described herein are also effective in thereduction of the supernatant volume of the original source of biologicalfluid. As shown in FIG. 12, supernatant can be reduced to approximately10% of its original volume (with the optional addition of washsolution). A further reduction the in the supernatant of FIG. 12 can beperformed by again concentrating the cells and removing additionalsupernatant (as shown in FIG. 13) such that the supernatant makes upapproximately 1% of the original supernatant volume in the source ofbiological fluid, or a 2-log reduction in the supernatant. Furtherreductions are also possible, thus making the system and methodsdescribed herein effective in reducing large culture volumes (e.g.,20-40 liters) down to a manageable volume for subsequent administration.

In another alternative application of the system and methods describedherein, it may be the supernatant that is the desired product. This maybe particularly applicable in the field of vaccine production, where itmay be desirable to remove the cellular components and retain thesupernatant (to produce a vaccine). In this embodiment, what wasreferenced to as the “waste” container (140, 140′, etc.) would, ineffect, become a product container.

Thus, an improved system for the sequential washing and processing ofbiological cells utilizing a series of disposable processing sets in asterile fashion is disclosed. The description provided above is intendedfor illustrative purposes only and is not intended to limit the scope ofthe invention to any specific method, system, or apparatus, or devicedescribed herein.

1-17. (canceled)
 18. A method for the sterile sequential treatment of abiological fluid comprising: (a) obtaining a reusable cell processingapparatus comprising a separator element for receiving a separationdevice for effecting the separation of biological fluid into two or morefractions; (b) obtaining a first disposable fluid circuit comprising aseparation device in fluid communication with a pre-attached firstproduct container and an access device for sterile connection to asource of biological fluid; (c) mounting said first disposableprocessing circuit onto said reusable cell processing apparatus; (d)sterilely joining a source container of biological fluid to said firstdisposable fluid circuit; (e) introducing said biological fluid fromsaid source container into said separation device to separate a desiredbiological cell component from a remainder of said biological fluid; (f)collecting said separated biological fluid component in said productcontainer; (g) transferring said collected biological fluid of saidproduct container to a pre-attached separation chamber; (h) removingsaid first disposable processing set from said reusable cell processingcircuit; (i) sterilely joining said separation chamber to a seconddisposable cell processing circuit, said second disposable processingcircuit comprising a separation device in fluid communication with apre-attached second product container and an access device for sterileconnection to a source of biological fluid; (j) mounting said seconddisposable processing circuit onto said reusable cell processingapparatus; (k) introducing the contents of a source container into saidseparation device to separate a desired biological fluid component froma remainder of said biological fluid; and (l) collecting a desiredseparated biological fluid fraction in said second product container ofsaid second disposable fluid circuit.
 19. The method of claim 18 furthercomprising: (a) transferring the collected fraction of said secondproduct container of said second disposable fluid circuit to apre-attached separation chamber; (b) sterilely joining said separationchamber to a third disposable cell processing circuit, said thirddisposable fluid circuit comprising a separation device in fluidcommunication with a pre-attached third product container and an accessdevice for sterile connection to a source of biological fluid; and (c)introducing a biological fluid from a source container into saidseparation device of said third disposable processing circuit toseparate a desired biological fluid component from a remainder of saidcontents.
 20. The method of claim 19 further comprising combining thecontents of said third product container with a treating agent.
 21. Themethod of claim 20 wherein said treating agent is combined with saidsecond product container contents in said second product container. 22.The method of claim 19 further comprising separating a desiredbiological fluid component from the contents of said second productcontainer and collecting said desired biological fluid component in athird product container.
 23. The method of claim 22 further comprisingtransferring said desired fluid component in said third productcontainer to a container set including two more containers. 24-26.(canceled)
 27. The method of claim 21 wherein said treating agent iscombined with a carrier solution prior to combination with said secondproduct. 28-35. (canceled)